This proposal is submitted in response to the RFA, ?Research answers to NCI's provocative questions?, in particular, question 10, which asks, How do microbiota affect the response to cancer therapies? Our preliminary data show that when tumor cells colonize the omentum, a fatty tissue in the peritoneal cavity, they trigger a profound Treg-mediated tolerogenic response that prevents tumor-specific CD8 T cells from clearing the tumor. Interestingly, this activity is specific for the omentum and involves a subset of Tregs known as visceral-adipose tissue (VAT)-associated Tregs. VAT-associated Tregs uniquely express the transcription factor PPARg and also express the ST2 component of the IL-33 receptor on their surface. These cells are found exclusively in adipose tisses and are not found in conventional lymhoid organs (like the spleen and lymph nodes) and are not found in peripheral sites. Importantly, VAT-associated Tregs are found in the omentum, which is the site of ovarian cancer metastasis. In additioin to showing that VAT-associated Tregs profoundly impair immunity to tumors that colonize the omentum, we also found that this activity is completely dependent on gut microbiota. As a result, VAT-associated Treg activity is impaired and anti-tumor immunity is restored in the omenta of germ-free mice. These results are surprising, since published data show that microbiota promote (rather than prevent) the anti-tumor effect of chemotherapy. These results were explained by the ability of chemotherapy to compromise the gut epithelium, allowing the translocation of microbiota and thereby triggering an IL-17 response, which facilitates the effects of chemotherapy on the clearance of tumors. Based on these data, the central hypothesis of this proposal is that the microbiota play opposing roles in promoting the immune suppressive Tregs under steady state conditions and by promoting the immune stimulatory Th17 responses following chemotherapy. Moreover, these types of responses are location dependent, with VAT-associated Tregs residing and responding primarily in fatty tissues like the omentum. Thus, therapy for ovarian cancer, which routinely metastasizes to the omentum, may have different outcomes than therapy to tumors in other locations. The experiments in this application test this hypothesis using a spontaneous mouse model of ovarian cancer and determine the mechanistic links between the gut microbiota and immunity in the peritoneal cavity using and ectopic model of ovarian cancer.